The statements in this section merely provide background information related to the present disclosure and may not constitute prior art.
Technology that can provide functional and structural imaging of tissues at a cellular level is of great importance in various fields. Specifically, in the field of clinical practice, histopathologic diagnosis, long considered the gold standard, is based on imaging thin, stained tissue specimens of biopsy or surgical resection, necropsy or autopsy-derived samples, a process that can require hours to multiple days to complete. Ideally, the histologic, genetic or phenotypic information would be attainable in vivo or very quickly after removal of a specimen. We will discuss several potential application areas for a rapid microscopy system, including surgical margin evaluation, biopsy quality control, rapid diagnosis, and rapid molecular characterization. These features are important for clinical pathology applications, but also in the biology, pharmacology and toxicology research settings. Additionally, the tissue sample may still be in-situ in a living organism, as with skin or oral mucosa, as long as it is accessible to appropriate imaging optics.
Surgical margin evaluation: Frozen-section evaluation of biopsies obtained during surgery can be routine part of clinical care but is fraught with difficulties. These include the time involved in orienting, embedding, freezing, cutting, staining, and viewing the resulting stained sections. This process can take 10 minutes or longer per specimen. Moreover, the quality of most of the frozen specimens may be less than optimal, and often lower than that of formalin-fixed paraffin-embedded (“FFPE”) specimens. The resulting delays and interpretation challenges limit the use of intra-operative biopsy or surgical margin evaluation. Consequently, if margin assessment is not done intra-operatively, additional surgeries may be required. For example, between 20 and 40% of breast cancer surgeries have to be revisited to remove residual cancer present at or near surgical margins; cancer deposits which ideally would have been detected during the original surgical procedure.
The need for frozen section replacement is well appreciated and many groups and companies have efforts in this area. Techniques include line-scanning confocal systems, wide-field OCT, multi-photon microscopy, as well as other non-imaging based approaches that include light scattering, spectroscopy, electrical impedance, and so on.
Biopsy quality control is another area that the present disclosure aims to address. It is important that biopsies, especially small, relatively non-invasive needle biopsies, contain the tissue of interest. For renal diagnosis, needle biopsies must contain glomeruli; for cancer diagnosis, of course the lesion must be properly sampled, and so on. In the case of samples that have to be partitioned for various purposes—histopathology, flow cytometry, nucleic acid extraction, for example, it is desirable that each aliquot of tissue has the cells or structures of interest. Moreover, it is important in some cases to have an estimate of what percentage of tumor verses stroma may be represented. Having a non-destructive method of rapidly examining the biopsy tissue to ensure that the appropriate content is present would be useful.
Rapid diagnosis of removed tissue samples is also important. Most tissues removed undergo conventional FFPE processing before definitive diagnoses are rendered, incurring delays of days or even weeks, depending on workflow. Having an ability to render diagnoses at the time of biopsy or surgery could decrease delays, diminish patient distress, encourage same-day clinical planning, and decrease overall costs.
Rapid molecular characterization of removed tissue samples is also important. Similarly, many molecular tests, such as immunohistochemistry or immunofluorescence for cancer markers or companion diagnostics are not ordered until after the initial tissue diagnosis, and incur additional days to weeks of delay. Having a microscope system that could provide morphological confirmation along with concurrent or fast but subsequent molecular staining could generate all the necessary tissue-based information on a same-day basis, which could be highly beneficial to the patient, and cost-saving to the provider.
Previous work by Lawrence Livermore National Laboratory and the University of California-Davis resulted in the development of a new imaging method to address these issues including applications in in-vivo imaging. A patent that describes subject matter resulting from this joint work is U.S. Pat. No. 7,945,077 to Demos et al., for “Hyperspectral Microscope for In-vivo Imaging of Microstructures and Cells in Tissues.” U.S. Pat. No. 8,320,650 to Demos, assigned to Lawrence Livermore National Security, LLC, and entitled “In-vivo Spectral Micro-Imaging of Tissue,” is also related to in-vivo imaging of microstructures and cells in tissues. These two patents are hereby incorporated by reference into the present disclosure. The techniques described in these two U.S. patents enable visualization of the tissue structure and organization at cellular scale in unprocessed tissue specimens. This imaging technology utilizes two physical mechanisms or characteristics. The first is the use of ultraviolet (UV) light that only superficially penetrates tissue. More specifically, the UV light only penetrates tissue on the order of a few micrometers to a few tens of a micrometer, depending on tissue type and wavelength. As a result, the fluorescence signal produced in this superficial tissue layer can be contained within the comparable thickness of the depth of field of the microscope. Oblique angle illumination was also used as means to limit the photon penetration depth for excitation at any given wavelength. The penetration depth can be defined in various ways such as the depth at which the amount of light dose reaching this depth is 1/e (or another predetermined fraction quantity such as 10%) of the incident amount of light.
The second main physical mechanism or characteristic is the use of native fluorophores within the cell compartments of the tissue being analyzed. There is sufficient variability in the concentration of native fluorophores (such as tryptophan, collagen, elastin, NADH) contained within cell compartments providing a natural “staining” method. In addition, images based on the emission of contrast agents can be attained and can be combined with those of native fluorophores to provide additional molecular information.
The methodology of the present disclosure offers several capabilities including: 1) the use of native tissue fluorescing biomolecules and exogenous dyes and labels for image acquisition; 2) short image acquisition times (on the order of milliseconds); and 3) facilitates incorporation into a wide range of instrumentation designs, including hand-held devices. Importantly, the methodology of the present disclosure is considerably less complex and less expensive than prior used technologies. These are significant advantages when compared to other emerging technologies.
In a clinical setting, consequently, there still exists a need for a system and methodology that reduces or eliminates the need for frozen section evaluation of biopsied human and animal tissue. More particularly, there is a need for a system and method which is well suited to intra-operative biopsy and/or surgical margin evaluation of freshly excised tissue samples, without requiring time-consuming and costly freezing and physical sectioning of relatively large tissue samples, and also which is well adapted to be used with conventional fluorescing stains and labels for further aiding evaluation, diagnosis and surgical margin analysis of a tissue sample. In the field of animal research, for basic understanding of body function, for studying the onset and progression of disease, in drug discovery and other areas, there is a need for instrumentation that can provide fast and inexpensive evaluation of a tissue specimen, and that obviates the need to perform conventional time-consuming, technically challenging and relatively expensive histopathology evaluations. The invention discussed here addresses a number of the aforementioned needs.